Oridonin Protected Mice From Experimental Autoimmune Encephalomyelitis By Targeting IL-2-IL-2R-Ras Signaling Pathway

Background and ObjectiveMultiple sclerosis(MS)is a chronic autoimmune disease of the central nervous system(CNS).The disease is prevalent in young and middle-aged people,especially women.MS has complicated clinical manifestations.Motor dysfunction is more common.In severe cases,it can cause paralysis,which is one of the main causes of neurological disability in young people.Experimental autoimmune encephalomyelitis(EAE)shares similar pathological characteristics and is a commonly used animal model to study multiple sclerosis.The pathogenesis of multiple sclerosis is not very clear,and in clinical,immunosuppressive agents and hormones was used to control the progress of the disease.However,the long-term use of immunosuppressants and hormones brings serious adverse reactions to patients.Chinese medicine,as immune modulators,can reduce the amount of hormones and immunosuppressive agents and antagonize their side effects.Oridonin,a tetracycline diterpene compound,is the main active ingredient of Rabdosia rubescens,and has various pharmacological effects such as anti-inflammatory and anti-tumor.It is reported that oridonin treatment can improve the clinical symptoms of experimental autoimmune neuritis.Previous studies have found that oridonin can prevent and treat EAE,but the specific molecular mechanism is still unclear.This study explored the molecular mechanism and target of oridonin in the treatment of EAE,which provided new ideas and theoretical basis for clinical treatment of MS.MethodsIn this study,C57BL/6 mice were subcutaneously immunized with MOGpeptides on the back of both sides to establish an EAE model.The mice were randomly divided into a control group(EAE group)and oridonin treatment group(ORI group).Oridonin(15 mg/kg/d)was injected intraperitoneally on days 3,5,and 7 after immunization.The EAE group was injected with the same amount of PBS.The incidence of mice in each group was observed daily.The therapeutic effect of oridonin on EAE was analyzed by clinical scores,histopathological changes,and CNS cell counts of the two groups of mice;the percentages of spleen T cells(CD4+and CD8+T cells),macrophages(CD11b+),B cells(CD 19+),and MOG-responsive CD4+T cells were measured by flow cytometry in two groups of mice to study cell subpopulations of oridonin;the ratio of Th1,Th2,Th17 and Treg cells were analyzed by flow cytometry and the cytokines IFN-y,IL-4,IL-17 and IL-10 were detected by ELISA of two groups of mice to explore the regulation effect of oridonin on T cell subsets;western blot was used to detect the expression of NF-κB/p65,p-p65,p-IκBα,ERK1/2,p-ERK1/2,c-fos,p-c-fos,c-jun,p-c-jun and p-mTOR proteins to study the effect of oridonin on NF-κB,MAPK and mTOR signaling pathways;calcineurin activity was analyzed by colorimetric method,NFAT expression was detected by flow cytometry,ELISA was used to detect the concentration of IL-2 and RT-PCR was used to detect the expression of IL-2 mRNA to study the effect of oridonin on calcineurin-NFAT signaling pathway;The Ras activity was detected by Ras activity detection kit and the expression of IL-2 receptor CD25 was analyzed by flow cytometry to study the effect of oridonin on Ras signaling pathways.Results1.Oridonin can treat EAE:After the immunization with MOG peptide,the mice in the control group all started to develop disease on the 8th day.Mice showed the initial symptom of tail weakness,followed by paralysis of the tail.On the 17th,it entered the peak of the disease,manifested as paralysis of both hind limbs and even involving the forelimbs,which could lead to death in severe cases.After oridonin treatment,the time of onset was significantly delayed.Mice started to develop disease on the 12th day.Mice were predominantly weak in the tail and did not involve hind limbs.In the EAE control group,CNS showed a large number of inflammatory cells infiltration.Compared with the EAE group,the number of CNS infiltrating mononuclear cells in the ORI group was significantly reduced.2.Oridonin inhibits CD4+T cell proliferation:Oridonin decreased the proportion of CD4+T cells in the spleen and CNS,but had no effect on the proportion of macrophages,CD8+ T and B cells.Oridonin can inhibit the proliferation of MOG-responsive CD4+T cells in the spleen.3.Oridonin regulates T cell subsets:Myelin-specific Th1 and Th17 cells are currently considered to be the main pathogenic cells of EAE.Oridonin treatment significantly inhibited Th1 and Th17 cell differentiation,but had no effect on Th2 and Treg cells.Oridonin treatment inhibited the secretion of inflammatory cytokines IFN-y and IL-17 and had no significant effect on the anti-inflammatory cytokines IL-4 and IL-10.4.Oridonin inhibits the activation of NF-κB,MAPK,and mTOR signaling pathways:NF-κB is an important inflammatory mediator and plays a key role in the inflammatory response and immune response process.The MAPK signaling pathway participates in multiple cellular programs such as cell proliferation and differentiation,and plays an important role in T lymphocyte activation and Thl and Th17 differentiation in vivo.The mTOR signaling pathway is closely related to T cell proliferation and regulates cell growth,metabolism and autophagy.Western blot results showed that oridonin can inhibit the phosphorylation of NF-κB,MAPK,and mTOR signaling pathway-related proteins.5.Oridonin has no effect on the calcineurin signaling pathway:The calcineurin-NFAT signaling pathway plays a key role in T cell proliferation and differentiation.Colorimetric and flow cytometry results showed that oridonin had no effect on calcineurin activity and NFAT transcription.However,ELISA and RT-PCR results showed that oridonin could up-regulate IL-2 expression.6.Oridonin targets IL-2-IL-2R-Ras signaling pathway to treat EAE:IL-2,also called T cell growth factor,is related to the proliferation and differentiation of lymphocytes.IL-2 interacts with the IL-2 receptor(CD25),which activates downstream Ras activation.Ras can regulate downstream signaling pathways of NF-κB,MAPK and mTOR.Pull down experiment showed that oridonin could inhibit Ras activity.Flow cytometry further proved that oridonin can inhibit the expression of IL-2 receptor CD25.ConclusionOridonin targets CD25 expression,inhibits IL-2/IL-2R-mediated Ras activation,inhibits NF-κB,MAPK,and mTOR signaling pathway activation,inhibits T cell proliferation,Th1 and Th17 cell differentiation and inflammation factor expression to treat experimental autoimmune encephalomyelitis.